Cell Line, System and Method for Optical-Based Screening of Ion-Channel Modulators

ABSTRACT

A variety of applications, systems, methods and constructs are implemented for use in connection with screening of ion-channel modulators. Consistent with one such system, drug candidates are screened to identify their effects on cell membrane ion channels and pumps. The system includes screening cells having light responsive membrane ion switches, voltage-gated ion switches and fluorescence producing voltage sensors. A chemical delivery device introduces the drug candidates to be screened. An optical delivery device activates the light responsive ion switches. An optical sensor monitors fluorescence produced by the voltage sensors. A processor processes data received from the optical sensor. A memory stores the data received from the optical sensor.

RELATED PATENT DOCUMENTS

This patent document claims the benefit, under 35 U.S.C. §119(e), ofU.S. Provisional Patent Application Ser. No. 60/955,116, entitled CellLine, System and Method for Optical-Based Screening of Ion-ChannelModulators and filed on Aug. 10, 2007; this patent document is fullyincorporated herein by reference.

This patent document also claims priority, as a CIP under 35 U.S.C.§120, to the following patent documents which are also individuallyincorporated by reference: U.S. patent application Ser. No. 11/651,422(STFD.150PA) filed on Jan. 9, 2007 and entitled, System for OpticalStimulation of Target Cells), which is a CIP of U.S. patent applicationSer. No. 11/459,636 (STFD.169PA) filed on Jul. 24, 2006 and entitled,Light-Activated Cation Channel and Uses Thereof which claims the benefitof U.S. Provisional Application No. 60/701,799 (STFD.169P1) filed Jul.22, 2005.

FIELD OF THE INVENTION

The present invention relates generally to systems and approaches forscreening drug candidates and more particularly to a cell line, systemand method for optically-based screening of the drug candidates withrespect to their effect on cellular ion channels.

BACKGROUND

Ion channels and ion pumps are cell-membrane proteins that control thetransport of positively or negatively charged ions (e.g., sodium,potassium and chloride) across the cell membrane. Ion channels play animportant part of various animal and human functions including signalingand metabolism. Ion-channel dysfunctions are associated with a widevariety of illnesses. For instance, diseases resulting from ion-channeldysfunctions in the central nervous system include anxiety, depression,epilepsy, insomnia, memory problems and chronic pain. Other diseasesresulting from ion-channel dysfunctions include cardiac arrhythmia, andtype II diabetes. Researchers are continually discovering diseasesassociated with ion-channel functionality.

Several drugs have been discovered to modify ion-channel functionality;however, the number of clinically approved drugs for restoringion-channel functionality is limited. A major bottleneck in thediscovery and development of new ion-channel drugs lies in the technicalchallenge of quickly, efficiently and cheaply screening drug candidatesto identify structures that affect ion-channel functionality. Commonscreening techniques use patch clamping to measure the voltage and/orcurrent in a cell. Micropipettes affixed to the cell membrane obtain themeasurement. For example, whole-cell configuration can be used tomonitor the functionality of the ion channels throughout the cell. Inthis manner, changes in voltage or current due to an introduced drug canbe monitored. Such methods require contact between the micropipette andthe cell. For this and other reasons, such techniques leave room forimprovement in their ability to screen drugs quickly, efficiently andcheaply.

These and other issues have presented challenges to screening of drugcandidates, including those affecting ion-channel function.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be more completely understood in consideration of thedetailed description of various embodiments of the invention thatfollows in connection with the accompanying drawings, in which:

FIG. 1A shows a block diagram of a system for optical drug screening,according to an example embodiment of the present invention;

FIG. 1B shows a specific system diagram of a large-format,quasi-automated system for drug screening in accordance with the presentmethodology, according to an example embodiment of the presentinvention;

FIG. 2 shows a system diagram of a small-format, fully automated drugscreening system which operates in accordance with the inventedmethodology, according to an example embodiment of the presentinvention;

FIG. 3A depicts the workings of an example of emitter/detector units,according to an example embodiment of the present invention;

FIG. 3B depicts the workings of another embodiment of emitter/detectorunits, according to an example embodiment of the present invention;

FIG. 4A depicts an electronic circuit mechanism for activating the LEDemitters used within the emitter/detector units, according to an exampleembodiment of the present invention;

FIG. 4B depicts an electronic circuit mechanism for light detection bythe emitter/detector units, according to an example embodiment of thepresent invention;

FIG. 5 shows a timeline for a sequence of events in the context of anexample screening process, according to an example embodiment of thepresent invention;

FIG. 6 illustrates an example of a layout of cell and drug sampleswithin the wells of a well-plate, according to an example embodiment ofthe present invention; and

FIG. 7 illustrates the context in which the disclosed invention may beemployed within a larger system that facilitates high-throughput drugscreening, according to an example embodiment of the present invention.

While the invention is amenable to various modifications and alternativeforms, specifics thereof have been shown by way of example in thedrawings and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

DETAILED DESCRIPTION

The present invention is believed to be useful for enabling practicalapplication of a variety of optical-based screening systems, and theinvention has been found to be particularly suited for use in systemsand methods dealing with identification of ion-channel modulating drugs.While the present invention is not necessarily limited to suchapplications, various aspects of the invention may be appreciatedthrough a discussion of various examples using this context.

Recently discovered techniques allow for stimulation of cells resultingin the rapid depolarization of cells (e.g., in the millisecond range).Such techniques can be used to control the depolarization of cells suchas neurons. Neurons use rapid depolarization to transmit signalsthroughout the body and for various purposes, such as motor control(e.g., muscle contractions), sensory responses (e.g., touch, hearing,and other senses) and computational functions (e.g., brain functions).Thus, the control of the depolarization of cells can be beneficial for anumber of different biological applications, among others includingpsychological therapy, muscle control and sensory functions. For furtherdetails on specific implementations of photosensitive bio-molecularstructures and methods, reference can be made to one or more of theabove-listed patent documents (by Karl Deisseroth et al.) which arefully incorporated herein by reference. These references discuss use ofblue-light-activated ion-channel channelrhodopsin-2 (ChR2) to causecalcium (Ca++)-mediated neural depolarization. Also discussed in one ormore of these references are other applicable light-activated ionchannels including, for example, halorhodopsin (NpHR) in which amberlight affects chloride (Cl−) ion flow so as to hyperpolarize neuronalmembrane, and make it resistant to firing. Collectively, theselight-sensitive proteins, serving to regulate membrane voltage using ionswitches that, when activated (or deactivated) in response to light,function as channels or pumps, are referred to herein aslight-responsive ion switches or light-activated membrane potentialswitches (LAMPS).

Consistent with one example embodiment of the present invention, asystem screens for ion-channel and ion-pump affecting compounds. Thesystem introduces one or more drug candidates that could either block orenhance the activity of ion-channels or ion-pumps to cells that weremade optically responsive by the addition of the above mentionedproteins (ChR2 and NpHR), for the purpose of screening the drugcandidates. Light triggers optically responsive ion channels in thecells causing a change in the voltage seen across the cell membrane. Thevoltage change stimulates voltage-gated ion channels in the cells whichwill then cause a change in ion concentrations that can be read asoptical outputs. These optical signals are detected and used todetermine what effect, if any, the drug candidates have on thevoltage-gated ion channels.

In addition to NpHR and ChR2, there are a number of channelrhodopsins,halorhodopsins, and microbial opsins that can be engineered to opticallyregulate ion flux or second messengers within cells. Various embodimentsof the invention include codon-optimized, mutated, truncated, fusionproteins, targeted versions, or otherwise modified versions of such ionoptical regulators. Thus, ChR2 and NpHR (e.g., GenBank accession numberis EF474018 for the ‘mammalianized’ NpHR sequence and EF474017 for the‘mammalianized’ ChR2(1-315) sequence) are used as representative of anumber of different embodiments. Discussions specifically identifyingChR2 and NpHR are not meant to limit the invention to such specificexamples of optical regulators. For further details regarding the abovementioned sequences reference can be made to “Multimodal fast opticalinterrogation of neural circuitry” by Feng Zhang, et al, Nature (Apr. 5,2007) Vol. 446: 633-639, which is fully incorporated herein byreference.

In one instance, the system allows for different drug candidates to bescreened without necessitating extensive setup between screenings. Forexample, an assay may be performed using optics both to stimulate theoptically responsive cells and to detect the effectiveness of the drug.The use of optics instead of manual contacts, e.g., using a whole-cellpatch clamp, can be particularly useful in increasing the throughput ofthe assay screening process. For instance, the time between screeningscan be reduced by minimizing or eliminating physical manipulationsotherwise necessary to stimulate or detect ion flow in the target cells.The cells can also be prepared prior to the screening process becausethe test equipment need only be optically coupled to the prepared cells.In another instance, throughput may be increased by screening a numberof different drugs simultaneously using, for example, an array of photodetectors and a corresponding array of modified cells exposed todifferent drugs.

Consistent with another embodiment of the present invention, anoptically-responsive cell line is created to screen for drugs thataffect the functionality of ion channels. The cell line includes cellsthat co-express optically responsive ion switches of Channelrhodopsin-2(ChR2) or NpHR, a voltage-gated Ca2+ channel and a hyperpolarizingchannel/pump (e.g., hERG or TASK1, that can lower the membrane voltageto a point where the voltage-gated Ca2+ channel will be in a closedstate). The system measures the concentration of Ca2+ using an indicatordye (e.g., Fura-2) or genetically encoded activity sensor. The abovementioned components are introduced to the cell line by standardliposomal transfection methods and the ChR2 related channel isstimulated using (blue) light; for further information in the regard,reference may be made to the patent documents cited herein and to thearticles cited supra. Time lapse images of light from the Ca2+ sensitiveportion of the system are taken and stored as data. A processor analyzesthe data to identify potential channel-affecting drugs. For instance,the processor may identify all chemicals that have concentrations ofCa2+ that do not fall within expected parameters (e.g., concentrationsthat exceed or are less than an expected range of concentrations).

In a specific instance, the cell line is derived from 293T cells byco-expressing ChR2 and a voltage-gated Ca2+ channel. The 293T cells (and293T cell line) are a variant of Human Embryonic Kidney (HEK) cells thatinclude the Simian vacuolating virus 40 (SV40) T antigen (see, e.g., N.Louis, C. Evelegh, F. L. Graham, Cloning and sequencing of theCellular-viral junctions from the human adenovirus type 5 transformed293 cell line, Virology., 233(2):423-9, Jul. 7, 1997; see also U.S. Pat.Nos. 5,939,320 to Littman, et al. filed Jun. 19, 1996, 6,790,657 to Aryafiled Jun. 28, 2001 and 6,489,115 to Lahue, et al. filed Dec. 3, 2002).Expression of the light-responsive ion channels, the voltage-gated ionchannels and the hyperpolarizing channels by the 293T cells may beaccomplished using appropriate transfection vectors.

More specifically, the cell lines may be derived from a stablehomogeneous cell line such as HEK293, NIH3T3, or CHO. Several genesresponsible for making different subunits of calcium channels have beenintroduced into the cell lines to provide functional calcium channelactivity. In addition to the calcium channel genes, an inward-rectifyingpotassium channel may be expressed to mimic the natural state of calciumchannels by maintaining a more hyperpolarized membrane potential(compared to the default resting membrane potential of HEK293, NIH3T3,or CHO cell lines). Also, a light-activated cation channelchannelrhodopsin-2 (ChR2) may be expressed to facilitate opticaldepolarization and subsequent activation of the calcium channels.Another option includes the expression of a light-activated chloridepump Natronomonas pharonis halorhodopsin (NpHR) to enable rapid opticalhyperpolarization of the cell membrane potential.

This cell line based approach is not limited to voltage-gated calciumchannels. For example, similar cell lines can be created forvoltage-gated sodium (e.g., Na_(v)1.1 through Na_(v)1.9), potassium(e.g., K_(v) such as hERG, TASK1, Shaker, or KvLQT1), or chlorideconducting channels/pumps (e.g., members of the CLC family of chloridechannels). The methods of introducing such genes into the cell line areknown in the art and may include, for example liposomal tranfection, orviral gene transfer. For further information in this regard, referencemay be made to one or more of the following references:

Warren Pear, Transient Transfection Methods for Preparation ofHigh-Titer Retroviral Supernatant, Supplement 68, Current Protocols inMolecular Biology, 9.11.1-9.11.18, John Wiley & Sons, Inc. (1996).

R. E. Kingston, C. A. Chen, H. Okayama, and J. K. Rose, Transfection ofDNA into Eukarotic Cells. Supplement 63, Current Protocols in MolecularBiology, 9.1.1-9.1.11, John Wiley & Sons, Inc. (1996).

R. Mortensen, J. D. Chesnut, J. P. Hoeffler, and R. E. Kingston,Selection of Transfected Mammalian Cells, Supplement 62, CurrentProtocols in Molecular Biology, 9.5.1-09.5.19, John Wiley & Sons, Inc.(1997).

H. Potter, Transfection by Electroporation, Supplement 62, CurrentProtocols in Molecular Biology, 9.3.1-9.3.6, John Wiley & Sons, Inc.(1996).

T. Gulick, Transfection using DEAE-Dextran, Supplement 40, CurrentProtocols in Molecular Biology, 9.2.1-9.2.10, John Wiley & Sons, Inc.(1997).

R. E. Kingston, C. A. Chen, H. Okayama, Transfection and Expression ofCloned DNA, Supplement 31, Current Protocols in Immunology (CPI),10.13.1-10.13.9, John Wiley & Sons, Inc.

Each of the above references is incorporated by reference.

These and other transfer vectors may be generated using various geneticengineering techniques. For instance, the transfer vectors may bederived from a provirus clone of a retrovirus, such as animmunodeficiency virus (e.g., HIV-1 or HIV-2, or SIV). For furtherdetails on the use of 293T cells and transfection thereof, reference canbe made to U.S. Pat. No. 6,790,657 (entitled, Lentivirus Vector System,to Arya), which is fully incorporated herein by reference.

In one embodiment of the invention, optical stimulation of the modifiedcells may be altered to determine specific properties of an introduceddrug candidate. For example, the intensity of the optical stimulus maybe modified to change the corresponding level of depolarization. Thelevel of desired depolarization can be tuned to further characterize theeffectiveness of the drug under test. In another example, the opticalstimulus may include rapid pulsing of the light. By correlating thetemporal relationship between the optical stimulus and the resultantdetected fluorescence, the drug may be further characterized in terms ofa kinetic response. Thus, the drug may be characterized for a variety ofdifferent aspects including, but not limited to, the steady state effecton ion concentrations, a change in the level of depolarization necessaryto trigger the voltage gated ion channels and the effect on repeateddepolarization.

In one embodiment, the system allows for simple calibration of theoptical stimulation and/or detection. The modified cells may beoptically stimulated prior to introduction of the drug candidate. Theion channel responsiveness is detected and recorded. The recorded valuesmay be used as a baseline for comparison to the ion channelresponsiveness of the same modified cells after the introduction of thedrug under test. The recorded values may also be used to modify theoptical stimulus or the sensitivity of the optical detector. Suchmodifications may be applied to an individual test sample or an array oftest samples. For such an array of test samples, each test sample may beindividually calibrated by adjusting the corresponding optical stimulus.Similarly, each corresponding photo detector may be individuallyadjusted.

FIG. 1A shows a basic block diagram of a system for screening forion-channel affecting drugs, according to an embodiment of theinvention. Optical control 104 communicates with database 102, opticalsource 106 and optical detector 109. Optical source 106 provides opticalstimulus to test sample 108. Test sample 108 includes the drug undertest, cells with optically responsive ion channels, and a voltage/ionindicator. In one instance, the indicator fluoresces in response tolight from optical source 106. Optical control 104 may also include areconfigurable readout, so that as different LAMPS and different LEIAsare used, the same control system can be readily adapted to eachparadigm. Optical detector 109 produces a signal responsive to suchflorescence, and optical control 104 receives the produced signal. Theoptical control 104 stores data obtained from the signal in database102. The information stored may include factors such as the intensity,duration and wavelength of the detected light. In a particular instance,the stored data can be compared against baseline data, where thebaseline data corresponds to data recorded prior to the introduction ofthe drug to the test sample 108. In another instance, optical source 106may vary the intensity, duration or other parameters related to thecontrol of optical source 106. These and other parameters may be storedin database 102.

It should be apparent that optical source 106 may be implemented using asingle light source, such as a light-emitting diode (LED), or usingseveral light sources. Similarly, optical detector 109 may use one ormore detectors and database 102 may be implemented using any number ofsuitable storage devices.

FIG. 1B shows a system diagram of a large-format, quasi-automated systemfor drug screening in accordance with a specific embodiment of theinvention. Control device 101 (e.g., a computer or control logic)controls various processes, and serves as the central point of systeminput/output functions. The environment may be maintained at anappropriate temperature, humidity, carbon dioxide level and ambientlight level within the walls of the climate control chamber 105, withthe help of one or more sensors 114 (e.g., thermostat, carbon dioxidesensor and humidity sensor), carbon dioxide and humidifier apparatus112, and heater 110. Multi-well tray 141 contains test wells 140 forholding cultured cells, drugs, and other ingredients needed for eachtest. Tray 141 rests upon X-Y-Z table 125, the movement of which iscarried out by table actuators 120, under control of computer 101. Xenonlamp 155 emits high-intensity white light 156, which is passed throughcolor filter 160. In the case that ChR2 is used for stimulating thecells within wells 140, color filter 160 is blue, causing blue light 161to exit the filter, and strike dichroic mirror 170. Blue light 161 thenpasses upward, through microscope objective lens apparatus 130, andthrough bottom of transparent tray 141. In this fashion, the contents ofwells 140, with their transparent undersides, are illuminated. When aseparate wavelength of light is required to stimulate a fluorescentlight-emitting indicator of cellular activity, a filter of theappropriate specification may be substituted for the previous filter160, causing light of the proper wavelength for this latter task to bepiped toward well 140. If the cells within well 140 have beenlight-sensitized, and if the drug being tested in each of these wellsdoes not suppress the process, a light-emitting indicator of cellularactivity (LEIA), which has also been added to each well or expressed bythe cells via genetic modification, will emit light in accordance withthe voltage change caused by the effect of the light. This secondwavelength of light, which may be much smaller in magnitude than thestimulation light, is collected by microscope turret 135, and will alsobe passed through dichroic mirror 175, onto the lens of (CCD) camera180.

Dichroic mirror 170 allows for upward reflection of both the wavelengthrequired to stimulate the optical gating of the membrane (e.g., blue forChR2), and the wavelength required by any LEIA used (e.g., ultravioletfor FURA-2). This dichroic mirror may be arranged to allow passage ofthe output spectrum of the LEIA (e.g., blue-green for FURA-2) withminimal reflection or absorption.

FIG. 2 is a system diagram of an automated-drug-screening system,according to an example embodiment of the invention. Emitter/detectorunits 250 make up the emitter/detector array 251. Emitter/detector array251 matches the number, size, and layout of the wells on tray 240. Trayholding device 225 permits tray swapping mechanism 220 to rapidly move anew tray into position once testing of a given tray has been completed.The entire process may be automated, and under the control of device201. Device 201 can be implemented using a computer, control logic,programmable logic arrays, discrete logic and the like. The introductionof the drug candidates under test can also be automated using a machinethat provides a reservoir for storing the drugs and a dispensing nozzlefor injecting the drugs into the tray. In a manner similar to that shownby FIG. 1, the environment within the walls of the climate controlchamber 205 may be maintained at an appropriate temperature, humidity,carbon dioxide level and ambient light level, with the help ofthermostat, carbon dioxide sensor and humidity sensor 214, carbondioxide and humidifier apparatus 212, and heater 210. The use ofmultiple stimulator/detector elements simultaneously and in parallel,can be particularly useful for augmenting the speed of the overallprocess. Low cost elements may be used to make multiple paralleldetectors (e.g., the components detailed below in description of FIGS.3A and 3B), the multiple parallel emitter/detector units may also bequite economically feasible.

FIG. 3A depicts the workings of emitter/detector units, such as thoseshown in FIG. 2, according to an example embodiment of the invention. AnLED stimulates light-sensitive ion channels of cells located within awell, and a photodiode detects the response of a LEIA. In thisembodiment, device 301 includes LED 310, which produces light pulses311, at the proper wavelength, pulse frequency and intensity, so as tostimulate light-sensitive transgenic cells 305 in culture within well306. In the case that ChR2 is the molecular target being used, bluelight of 1-10 mW/mm2 is generally appropriate. Due to the presences ofan LEIA (e.g., a voltage-sensitive dye or a calcium dye), light 316 isreturned from cells 305, and is detected by photodiode 315. In the casethat RH 1691 being used, red light is fluoresced and detected byphotodiode 315. In the absence of cellular depolarization, nofluorescence is detected by photodiode 315. Other light detectingtechnologies may also be used instead of a photodiode includingphototransistors, and CCD elements.

The combination of photostimulation with optical imaging techniques ofLEIAs may be useful for a number of different reasons. For example,photostimulation may simplify the study of excitable cells by reducingthe need to use mechanical electrodes for stimulation. Severalcommercially available LEIAs are suitable for photogrammetricallyindicating the activation of electrically excitable cells. One such LEIAis calcium dye Fura-2, which may be stimulated with violet/ultravioletlight around 340 nm, and whose fluorescent output is detectable asblue-green light around 535 nm. Another example is voltage sensitive dyeRH 1691, which may be stimulated with green light at about 550 nm, andwhose fluorescent output is detectable as red light at about 70 nm.Another example is voltage sensitive dye di-4-ANEPPS, which isstimulated by blue light at about 560 nm, and whose fluorescent outputis detectable as red light at about 640 nm.

FIG. 3B depicts the workings of another embodiment of theemitter/detector units shown in the FIG. 2, in which multiple effectsare tested within the context of a single well. For example, the cells355 in the wells 356 may express both ChR2 and NpHR, and hence besensitive to both the depolarizing effects of blue light, and thehyperpolarizing effects of amber light. Device 351 includes LED 360,which is used for the stimulation of the targeted ion channel or pump(e.g., ChR2) of light-sensitive transgenic cells 355. Additional LED 375may be used to stimulate a second targeted ion channel or pump (e.g.,NpHR). Yet another LED 380 may be used to stimulate a voltage sensitivedye (e.g., RH1691 or calcium dye, such as Fura-2). Each LED may bearranged to output specific wavelengths and intensities for stimulus ofrespective targeted compounds. In one instance, an LED may affect morethan one target, depending upon the specific sensitivities of eachcompound used. Photodiode 365 detects the fluorescence of a selectedvoltage dye, while photodiode 370 is sensitive to the spectrumfluoresced by a selected calcium dye. The use of multiple LEDs for thesame cell allows for the stimulation of LEIAs at different wavelengths.Multiple LEDs may also be used to detect different light wavelengthsemitted by the LEIA.

FIG. 4A depicts an electronic circuit mechanism for activating the LEDemitters used within the emitter/detector units, according to an exampleembodiment of the invention. Control device 401 generates a “light onsignal” 402 to transistor base 405. This “light on signal “402 willremain on for the duration of a light flash desired, or alternativelymay turn on and off in order to produce rhythmic light flashes at aspecified frequency. Light on signal 402 permits (conventional) currentto flow from power source 410, through resister 411, and throughtransistor collector 407 and transistor emitter 412, to ground 413.Current is also thereby permitted to pass through resistor 415, and intoLED 420. LED 420 emits light 421, which falls upon well 425. In aparticular instance, the transistor functions as transconductanceamplifier of signal 402. In this manner, light of the appropriatewavelength, intensity and frequency is delivered to cells within thewell 425, so as to cause them to stimulate the particular ion channel(e.g., ChR2) or pump (e.g., NpHR), or other photoactive membranestructure being used to regulate the activity of electrically excitablecells. Various other circuits are also possible. For example, othercircuits can be used in place of circuit 406 to control LED 420including, but not limited to, replacing the transistor with anoperational amplifier, a field-effect-transistor, a resistor dividernetwork, transistor-transistor logic, push-pull driver circuits andswitches.

FIG. 4B depicts an example electronic circuit mechanism for lightdetection by the emitter/detector units, according to one embodiment ofthe invention. Control device 450 may (optionally, depending uponspecific implementation) provide power to photodiode 455. Photodiode 455receives fluoresced (emitted) light 456 from the LEIA on the cellswithin well 457. The received light results in an output signal. Thisoutput passes through resistor 460, and is input to Schmitt triggeredhex inverter 470, which conditions the signal, providing a clean “high”or “low value” to be input to computer 450.

Operation of the photodetector is shown in photovoltaic mode, but theelement may also be used in the photoconductive mode of operation. Ofcourse, many other light-detection devices and methods may also be used,including phototransistors, photothyristors, and charged-coupled device(CCD) elements, or arrays of elements.

Alternatively, the 4B circuit can be used without Schmitt-triggered hexinverter 470, permitting a continuum of signal intensities to betransmitted directly to an analog input to computer 450 or to ananalog-to-digital converter. Various other signal conditioning circuitsare also possible.

FIG. 5 shows a sequence of steps using the embodiment shown in FIGS. 2,3 and 4, in the context of projected high-throughput process time course500 and in accordance with one embodiment of the invention. In step 505,light of the appropriate wavelength and intensity for the targeted ionchannel is flashed—in this case for approximately 3 seconds.Concurrently, a LEIA stimulation flash 510 may optionally be triggered,depending upon the specific voltage or calcium dye, etc. being used.This LEIA compound may have been previously added to the well, or may be(artificially) genetically imparted upon the cells such that thechemical is produced/expressed by the cells. In step 515, the lightsignal produced by the LEIA is detected by the photodetector element(e.g. photodiode). For example, RH1691, fluoresces red light at about 70nm.

In step 520, the signal resulting from the impingement of light onto thephotodetector element is sent back to the computer. This may be a binary(e.g. “high” versus “low” signal intensity), or may be graded to reflecta continuum of activation levels. In the case that multiplephotodetectors are used to determine energies at different wavelengths,the individual readings of these photodetectors may be logged inparallel or in sequence for appropriate interpretation in a later stageof the automated process. In step 530, the system calls for the nexttray to be placed by the automated system. The next tray is moved intoposition at step 535 and the process may be repeated until all trays ina batch have been processed.

The level of light fluoresced is typically much lower than that requiredto optically stimulate a cell via light-sensitive ion channels or pumps.For example, ChR2 may require blue light of 1-10 mW/mm2 or more in orderto robustly depolarize cells. RH 1691 may require approximately 0.1mW/mm2 to stimulate it. Given that RH1691 shows significant sensitivityto blue light, (peak sensitivity is at the blue-green wavelengths),RH1691 is adequately stimulated by the same pulse used to stimulateChR2, but emits light upon depolarization at a power of only on theorder of 0.001 mW/mm2. This small amount of output light would bedifficult to distinguish from the comparatively massive blue pulse usedto stimulate ChR2, even if efficient filters were used in front of thedetectors. Fortunately, temporal differences between the ChR2stimulation (with simultaneous LEIA stimulation), and the fluorescentoutput of depolarized cells can be used to distinguish the lightsources. For instance, the dye-based fluorescence may continue for a fewseconds after the delivery of the depolarization pulse and the resultantaction potential. Thus in some instances, such as a non-fluorescent LEIAor a luminescent activity dye, a separate stimulation flash is notrequired.

The amount of time allotted for light delivery may vary, and depends onfactors including the level of light-gated ion channel/pump expression,and the density and characteristics of other ionic channelcharacteristics of that cell population. The amount of time allotted forlight receipt may vary, and depends upon factors including the degree ofaccuracy required for the screening session. The amount of time allottedfor well-plate (tray) changing may vary, and depends upon factorsincluding the mechanical speed of the automated apparatus. If fastneurons are used as the cells being tested, the cellular stimulation andLEIA detection process may be accomplished in milliseconds.

In an example process, a 293T cell line expressing TASK-1 (to simulatethe natural hyperpolarized membrane potential of neurons), ChR2 (toinduce depolarization of the cell membrane), and the L-type calciumchannel are used. Whole-cell patch clamping experiments show that themembrane of the modified 293T cell line is hyperpolarized to the pointwhere the L-type calcium channels are closed. The cells are stimulatedfor 5 seconds with continuous blue light (470 nm) to activate ChR2.ChR2-mediated depolarization opens the co-expressed voltage-gatedcalcium channels. Upon ChR2 illumination, a strong calcium influx isrecorded using a genetically-encoded calcium dye indicator, whichfluoresced light with cellular depolarization. Nimodopine, a well-knownL-type calcium channel blocker, abolishes the calcium influx- and hencethe fluoresced signal when applied to the cells for 10 minutes. Thisdata demonstrates the effectiveness of the system described herein.

The process above may be repeated under varying conditions. For example,a given set of cells may be tested with no drug present, andsubsequently with one or more drugs present. The response ofelectrically-excitable cells under those conditions may be therebydocumented, compared and studied. If the invention is implemented withat least one emitter/detector for each well on a tray and at least twoconcurrently operating devices, continuous operation may be maintainedfor extended periods of time.

FIG. 6 illustrates an example of a layout of cell and drug sampleswithin the wells of a well-plate which is suitable for use within anembodiment of the invention. In this figure, well-plate 601 (alsoreferred to herein as a “tray” contains wells 605 (examples), which areorganized into columns 625, labeled with numbers 1-12 and rows 620,labeled with letters A-H. More specifically, an example column and roware defined by 610 and 615 respectively.

As an example of a functional layout of contents introduced into thesewells, rows A-H of a single plate might be used for the testing of twodifferent drugs. To represent a baseline condition, column 1 mightcontain optically gated cells, an endogenous or exogenous LEIA, but nodrug. Columns 2-6 might be used for five different concentrations ofDrug X, one concentration level per column. Likewise, columns 7-11 mightbe use for five different concentrations of Drug Y, one concentrationper column. Column 12, while fully usable, is left unused in thisparticular example.

Variables in the various wells might include the type of cell beingtested, the type of ion channel being tested for, the type of drugplaced in the cell, the concentration of the drug placed in the well,the specific LETA used, and the optical gating stimulation parameters(e.g. wavelength, intensity, frequency, duration) applied to the cellsin that well.

FIG. 7 illustrates the context in which the disclosed invention may beemployed within a larger system which facilitates high-throughput drugscreening. Well-plate 706 contains wells 705. These are carried forwardby conveyer 720, which may be a device such as a conveyor belt, robotictransporter or other delivery mechanism. Pipettes 710 are held in arrayby robotic member 715, and serve to inject the proper number of culturedcells and media into wells 705. Subsequently, well-plate 706 is moveddown conveyer 720, where robotic member 725, analogous to robotic member715 and also containing pipettes, injects the proper amount of a LEIAinto wells 705. Conveyer 720 then brings well-plate 705 into screeningchamber 730. An emitter/detector apparatus, such as those described inconnection with FIG. 2, FIG. 3A, FIG. 3B, FIG. 4A, and FIG. 4B, islocated within chamber 730. Additionally, portions of the processesdescribed in FIG. 5 may occur within this chamber. Subsequently,well-plates 735 is moved out of screening chamber 730 by conveyor 740,and discarded at 745. In an alternative embodiment, one or more roboticdevices may move pipettes 710, screening chamber 730, etc. to thelocations of well-plate 706, rather than vice-versa.

Consistent with the above discussion, example screening methods couldinclude the collection of multiple data points without having to switchsamples. Because control over the samples is reversible in the samesample preparation by simply turning the activating light on and offwith fast shutters, the same samples can be reused. Further, a range ofpatterns of stimulation can be provided to the same cell sample so thattesting can be performed for the effect of drugs without concern withregards to differences across different sample preparations. Bymodulating the level of excitation (e.g., by ramping the level from nolight to a high or maximum intensity), the effect of the drug across arange of membrane potentials can be tested. This permits for theidentification of drugs that are efficacious during hyperpolarized,natural, or depolarized membrane potentials.

The cell lines described herein may be a particularly useful fordetailed characterization of drug candidates in a high-throughputmanner. Optical control is relatively fast, thereby allowing for thetesting the drug's activity under more physiological forms ofactivation. For example, different frequencies of depolarization and/orhyperpolarization may be used to determine how a drug interacts with thechannel under physiological forms of neural activity. In some instances,the process may be accomplished without the application of expensivechemical dyes to the cell lines.

In conjunction with the various properties discussed herein, the use ofvarious embodiments of the invention may be particularly useful forimproving screening throughput by eliminating the need for cumbersomemechanical manipulation and liquid handling. Various embodiments mayalso be useful for repeatable the screening assay using the samesamples, reducing screening cost by eliminating the need forchemically-based fluorescence reports, producing high temporal precisionand low signal artifact (due to the optical nature of the voltagemanipulation), modulating the level of depolarization by attenuating thelight intensity used for stimulation, and ascertaining the kinetics ofthe drug's modulation on the ion channel through the use of pulsed lightpatterns.

The various embodiments described above are provided by way ofillustration only and should not be construed to limit the invention.Based on the above discussion and illustrations, those skilled in theart will readily recognize that various modifications and changes may bemade to the present invention without strictly following the exemplaryembodiments and applications illustrated and described herein. Forinstance, such changes may include the use of digital logic ormicroprocessors to control the emitted light. Such modifications andchanges do not depart from the true spirit and scope of the presentinvention, which is set forth in the following claims.

1. A genetically modified cell line, wherein the genetically modifiedcell line is genetically modified to express: a voltage-gated ionchannel; and a light-responsive ion switch that mediates depolarizationfrom activation of the voltage-responsive ion channels.
 2. The cell lineof claim 1, wherein the ion switch is a ChR2 ion channel.
 3. The cellline of claim 1, wherein the ion switch is an NpHR ion pump.
 4. The cellline of claim 1, wherein the voltage-gated ion channel is a Ca2+channel.
 5. The cell line of claim 1, wherein the modified cell line isfurther genetically modified to express an ion indicator.
 6. The cellline of claim 5, wherein the ion indicator is a calcium indicator.
 7. Asystem for screening drug candidates to identify their effects on cellmembrane ion channels and pumps, comprising: recombinant cellsgenetically modified to express light responsive membrane ion switches,wherein the cells comprise voltage-gated ion switches and fluorescenceproducing voltage sensors; a chemical delivery device for introducingthe drug candidates to be screened; an optical delivery device toactivate the light responsive ion switches; an optical sensor to monitorfluorescence produced by the voltage sensors; a processor to processdata received from the optical sensor; and memory for storing the datareceived from the optical sensor.
 8. The system of claim 7, wherein thelight responsive membrane ion switches are ChR2 ion channels.
 9. Thesystem of claim 7, wherein the light responsive membrane ion switchesare NpHR ion pumps.
 10. The system of claim 7, wherein the voltage-gatedion switches are Ca2+ channels.
 11. The system of claim 7, wherein thefluorescence producing voltage sensors are genetically-encoded calciumdye indicators.
 12. The system of claim 7, further comprising an arrayof wells each containing a portion of the screening cells and an arrayof optical sensors for detecting light from respective wells from thearray of wells. 13-19. (canceled)